Inhibitors of dipeptidyl peptidase IV

ABSTRACT

Compounds according to general formula (1), wherein R 1  is H or CN, X 1  is S, O, SO 2  or CH 2 , X 2  is O, S or CH 2 , X 3  is NR 5  or a carbonyl or thiocarbonyl group and R 4  is R 6 R 7 N, R 8 (CH 2 ) q OC(═O), R 8 (CH 2 ) q OC(═S), R 6 R 7 NC(═O), R 6 R 7 NC(═S); R 8 (CH 2 ) q C(═O), R 8 (CH 2 ) q C(═S) or R 8 (CH 2 ) q SO 2 , m is 1-3 and n is 0-4 are new. The compounds of the invention are inhibitors of dipeptidyl peptidase IV. Pharmaceutical compositions of the compounds of the invention, or pharmaceutically acceptable salts thereof, are useful in the treatment of,inter alia, type 2 diabetes.

[0001] The present invention relates to a series of novel compounds thatare inhibitors of the enzyme dipeptidyl peptidase IV, to pharmaceuticalcompositions comprising these inhibitors, and the use of suchcompositions in the treatment of human diseases.

BACKGROUND

[0002] The enzyme dipeptidyl peptidase IV, herein abbreviated DP-IV (andelsewhere as DAP-IV or DPP-IV) and also known by the classificationEC.3.4.14.5, is a serine protease that cleaves the N-terminal dipeptidefrom peptides that begin with the sequence H-Xaa-Pro (where Xaa is anyamino acid, although preferably a lipophilic one, and Pro is proline).It will also accept as substrates peptides that begin with the sequenceH-Xaa-Ala (where Ala is alanine). DP-IV was first identified as amembrane-bound protein. More recently a soluble form has beenidentified.

[0003] Initial interest in DP-IV focussed on its role in the activationof T lymphocytes. DP-IV is identical to the T cell protein CD26. It wasproposed that inhibitors of DP-IV would be capable of modulating T cellresponsiveness, and so could be developed as novel immunomodulators. Itwas further suggested that CD26 was a necessary co-receptor for HIV, andthus that DP-IV inhibitors could be useful in the treatment of AIDS.

[0004] Attention was given to the role of DP-IV outside the immunesystem. It was recognised that DP-IV has a key role in the degradationof several peptide hormones, including growth hormone releasing hormone(GHRH) and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2). Since GLP-1is known to have a potentiating effect on the action of insulin in thecontrol of post-prandial blood glucose levels it is clear that DP-IVinhibitors might also be usefully employed in the treatment of type IIdiabetes and impaired glucose tolerance. At least two DP-IV inhibitorsare currently undergoing clinical trials to explore this possibility.

[0005] Several groups have disclosed inhibitors of DP-IV. While someleads have been found from random screening programs, the majority ofthe work in this field has been directed towards the investigation ofsubstrate analogs. Inhibitors of DP-IV that are substrate analogs aredisclosed in, for example, U.S. Pat. No. 5,462,928, U.S. Pat. No.5,543,396, WO95/15309 (equivalent to U.S. Pat. No. 5,939,560 and EP0731789), WO98/119998 (equivalent to U.S. Pat. No. 6,011,155),WO99/46272 and WO99/61431. The most potent inhibitors are aminoacylpyrrolidine boronic acids, but these are unstable and tend to cydise,while the more stable pyrrolidine and thiazolidine derivatives have alower affinity for the enzyme and so would require large doses in aclinical situation. Pyrrolidine nitrites appear to offer a goodcompromise since they have both a high affinity for the enzyme and areasonably long half-life in solution as the free base. There remains,however, a need for inhibitors of DP-IV with improved properties.

BRIEF DESCRIPTION OF THE INVENTION

[0006] The present invention relates to a series of inhibitors of DP-IVwith improved affinity for the enzyme. The compounds can be used for thetreatment of a number of human diseases, including impaired glucosetolerance and type II diabetes. Accordingly, the invention furtherrelates to the use of the compounds in the preparation of pharmaceuticalcompositions, to such compositions per se, and to the use of suchcompositions in human therapy. The compounds of the invention aredescribed by general formula 1.

[0007] In general formula 1, X¹ is selected from —S—, —O—, —SO—, —SO₂—and —CH₂—; X² is selected from —O—, —S—, —NH— and —CH₂—; X³ is either—NR⁵— or a >C═O or >C═S group; R¹ is either H or —CN; R² and R³ areindependently selected from H and lower alkyl, or together may be—(CH₂)_(p)—; R⁴ is R^(4A) when X³ is —NR⁵— and R^(4B) when X³ is >C═Oor >C═S; R^(4A) is selected from R⁶R⁷NC(═O), R⁶R⁷NC(═S);R⁸(CH₂)_(q)C(═O), R⁸(CH₂)_(q)C(═S), R⁸(CH₂)_(q)SO₂, R⁸(CH₂)_(q)OC(═S)and R⁸(CH₂)_(q)OC(═O); R^(4B) is R⁶R⁷N; R⁵ is H or lower alkyl; R⁶ andR⁷ are each independently R⁸(CH₂)_(q) or together they are—(CH₂)₂—Z—(CH₂)₂—; R⁸ is selected from H, alkyl, optionally substitutedaryl, optionally substituted aroyl, optionally substituted arylsulphonyland optionally substituted heteroaryl; Z is selected from a covalentbond, —(CH₂)_(r)—, —O—, —SO_(t)— and —N((CH₂)_(q)R⁸)—; n is 0-4; p is2-5; q is 0-3; r is 1 or 2; and t is 0-2.

DETAILED DESCRIPTION OF THE INVENTION

[0008] In a first aspect, the present invention comprises a series ofnovel compounds that are inhibitors of the enzyme DP-IV and are usefulfor the treatment of certain human diseases. The compounds are describedby general formula 1.

[0009] In general formula 1, X¹ is a divalent group selected from asulphur atom (—S—), an oxygen atom (—O—), a sulphinyl group (—SO—), asulphonyl group (—SO₂—) and a methylene group (—CH₂—). X² is a divalentgroup selected from an oxygen atom (—O—), a sulphur atom (—S—) and amethylene group (—CH₂—). X³ is either a substituted imino group (—NR⁵—)or a carbonyl (>C═O) or thiocarbonyl (>C═S) group.

[0010] R¹ is either a hydrogen atom (H) or a nitrile group (—CN).

[0011] R² and R³ may each independently of the other be a hydrogen atomor a lower alkyl group, or together they may be a chain of between twoand five methylene units (—(CH₂)_(p)— where p is in the range 2-5) so asto form, with the carbon atom to which they are attached, a three, four,five or six-membered ring. The value of m may be 1, 2 or 3. When m isgreater than 1 then each CR²R³ unit may be the same or different. Forexample, when m is 2 then (CR²R³)₂ may be CH₂CH₂, CH₂C(Me)₂, C(Me)₂CH₂and the like.

[0012] The nature of R⁴ depends on the identity of X³, such that the twogroups are linked by an amide (CO—N), thioamide (CS—N) or sulphonamide(SO₂—N) bond. So, when X³ is a substituted imino group (—NR⁵—) then R⁴is R^(4A), where R^(4A) is selected from carbamoyl groups (R⁶R⁷NC(═O)),thiocarbamoyl groups (R⁶R⁷NC(═S)); optionally modified acyl groups(R⁸(CH₂)_(q)C(═O)), optionally modified thioacyl groups(R⁸(CH₂)_(q)C(═S)), sulphonyl groups (R⁸(CH₂)_(q)SO₂), optionallymodified (alkyl or aryloxy)carbonyl groups (R⁸(CH₂)_(q)OC(═O)) andoptionally modified (alkyl or aryloxy)thiocarbonylgroups(R⁸(CH₂)_(q)OC(═S)). As used herein, the term “optionallymodified” is taken to indicate that some embodiments of R⁸ are beyondthe scope of the terms “alkyl”, “acyl” and “aryl”. The scope of thedefinition of R^(4A) is determined by the scope of the definition of R⁸.Alternatively, when X³ is a carbonyl (>C═O) or thiocarbonyl (>C═S) groupthen R⁴ is R^(4B), where R^(4B) is a substituted amino group (R⁶R⁷N).

[0013] R⁵ is a hydrogen atom (H) or a lower alkyl group. Preferably, R⁵is H.

[0014] R⁶ and R⁷ may each independently of the other be R⁸(CH₂)_(q).Alternatively, they may together be a group —(CH₂)₂—Z¹—(CH₂)₂— or—CHR⁹—Z²—CH₂—CR¹⁰—. Here Z¹ is a covalent bond, a methylene orethylidene group (—(CH₂)_(r)— where r is 1 or 2), an oxygen atom (—O—),a sulphur or oxidised sulphur atom (—SO_(t)— where t is zero, 1 or 2) ora substituted imino group (—N((CH₂)_(q)R⁸)—), such that the group NR⁶R⁷is a pyrrolidine, piperidine, perhydroazepine, morpholine, optionallyoxidised thiomorpholine or substituted piperazine ring. Z² is anortho-phenylene moiety (—C₆H₄—), such that the group NR⁶R⁷ is atetrahydroisoquinoline.

[0015] R⁸ is selected from a hydrogen atom (H), a lower alkyl group, abenzo-fused lower cycloalkyl group (such as an indanyl group), an acyl(lower alkyl-CO) group, a di(lower alkyl)amino group, a di(loweralkyl)carbamoyl group, an N-(lower alkyl)piperidinyl group, anoptionally substituted α-alkylbenzyl group, an optionally substitutedphenyl, naphthyl or heteroaryl group, and an optionally substitutedaroyl (aryl-CO) or arylsulphonyl (aryl-SO₂) group. In the foregoing,suitable optional substituents are lower alkyl, aryl which may befurther substituted with one or more methyl or trifluoromethyl groups,hydroxy, lower alkyloxy, lower alkylsulphonyl, acyl, perfluoroacyl,amino, lower alkylamino, di(lower alkyl)amino, aminoalkylene, fluoro,chloro, bromo, trifluoromethyl, nitro, cyano, carbamoyl, carboxy andlower alkyloxycarbonyl groups. In addition, two adjacent substituentsmay be linked so as to form a ring fused to the parent aryl orheteroaryl ring.

[0016] R⁹ and R¹⁰ are independently selected from hydrogen, carbamoyl,hydroxymethyl and cyanomethyl groups.

[0017] The integer n is selected from the range zero to 4, and q isselected from the range zero to 3.

[0018] Certain compounds are specifically excluded from the scope of thepresent invention. When X² is methylene, X³ is NH and R⁴ isR⁸(CH₂)_(q)O(CO), with q=1, then R⁸ may not be unsubstituted phenyl orphenyl substituted with a nitro group. It is generally preferred thatwhen X² is methylene, X³ is NH, R⁴ is R⁸(CH₂)_(q)O(CO), q is 1 and R⁸ isa substituted phenyl group then the substituent or substituents shouldbe selected from chloro, methoxy and trifluoromethyl groups.

[0019] In the context of the present disclosure, the term lower alkyl,either by itself or in such combinations as lower alkyloxy, is intendedto comprise linear, branched and cyclic saturated hydrocarbon groups ofbetween one and six carbon atoms. Examples of lower alkyl groupsinclude, but are not limited to, methyl, ethyl, isopropyl, tert-butyl,neopentyl, cyclohexyl, cyclopentylmethyl, 2-(cyclopropyl)ethyl,3,3-dimethylcyclobutyl and bicyclo[3.1.0]hexyl.

[0020] The term heteroaryl includes monocyclic five- and six-memberedring aromatic groups with one or two heteroatoms, which are selectedfrom nitrogen, oxygen and sulphur. Thus, heteroaryl groups includepyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl,imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl and isoxazolyl.Heteroaryl further includes the benzofused derivatives of these rings,such as quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl,phthalazinyl, cinnolinyl, indolyl, isoindolyl, benzothiazolyl and thelike, and bicyclic groups formed by the fusion of two such monocyclicheteroaromatic groups.

[0021] The term aryl includes phenyl, naphthyl and heteroaryl.

[0022] The compounds of general formula 1 have at least one stereogeniccentre and so can exhibit optical isomerism. All such isomers, includingenantiomers, diastereomers and epimers are included within the scope ofthe invention. Furthermore, the invention includes such compounds assingle isomers and as mixtures, including racemates. Certain compoundsaccording to general formula 1, including those with a heteroaryl groupwhich carries a hydroxy or amino substituent, can exist as tautomers.These tautomers, either separately or as mixtures, are also consideredto be within the scope of the invention.

[0023] The compounds according to general formula 1 have at least onebasic functional group. They can therefore form addition salts withacids. Those addition salts that are formed with pharmaceuticallyacceptable acids are included within the scope of the invention.Examples of suitable acids include acetic acid, trifluoroacetic acid,citric acid, fumaric acid, benzoic acid, pamoic acid, methanesulphonicacid, hydrochloric acid, nitric acid, sulphuric acid, phosphoric acidand the like.

[0024] Certain compounds according to general formula 1 have an acidicgroup and so are able to form salts with bases. Examples of such saltsinclude the sodium, potassium and calcium salts, which are formed by thereaction of the acid with the corresponding metal hydroxide, oxide,carbonate or bicarbonate. Similarly, tetra-alkyl ammonium salts may beformed by the reaction of the acid with a tetra-alkyl ammoniumhydroxide. Primary, secondary and tertiary amines, such astriethylamine, can form addition salts with the acid. A particular caseof this would be an internal addition salt formed between an acidicgroup and the primary amine group of the same molecule, which is alsocalled a zwitterion. Insofar as they are pharmaceutically acceptable,all these salts are included within the scope of the invention.

[0025] In a preferred embodiment of the invention R¹ is a nitrile group.Within this embodiment, it is preferred that the stereochemistry of thenitrile group is as shown in general formula 2.

[0026] According to the standard terminology, this is the Sconfiguration when X¹ is methylene but the R configuration when X¹ issulphur, oxygen, sulphinyl or sulphonyl.

[0027] In another preferred embodiment, the stereochemistry at thecentre adjacent to the primary amine is as shown in general formula 3.This is the S configuration when X² is an oxygen atom or a methylene orimino group, and the R configuration when X² is a sulphur atom.

[0028] Within this embodiment, it is more preferred that R¹ should be anitrile group, and more preferred still that it should have the absoluteconfiguration depicted in general formula 4.

[0029] In another preferred embodiment of the invention, m is 1. Morepreferably m is 1 and R² and R³ are independently hydrogen atoms ormethyl groups. When X² is a methylene group, it is more preferred thatR² and R³ both be hydrogen. When X² is an oxygen atom, it is morepreferred that one of R² and R³ be hydrogen and the other a methylgroup. When X² is a sulphur atom, it is more preferred that both R² andR³ be methyl groups.

[0030] In another preferred embodiment, X¹ is either S or methylene.More preferably, X¹ is S and R¹ is H, or X¹ is methylene and R¹ is CN.

[0031] In another preferred embodiment, X³ is NH. More preferably, X³ isNH, m is 1, R² and R³ are both H, X² is methylene and n is 1 or 2.

[0032] In another preferred embodiment, R⁴ is R⁶NHCO or R⁸CO and R⁸ isan optionally substituted heteroaryl group. More preferably, R⁸ is anunsubstituted heteroaryl group, or a heteroaryl group substituted withone or two groups chosen from lower alkyl, lower alkyloxy, fluoro,chloro and trifluoromethyl groups.

[0033] In another preferred embodiment, X³ is CO and R⁴ is R⁸NH. Morepreferably R⁸ is an optionally substituted heteroaryl group. Morepreferably still, R⁸ is an unsubstituted heteroaryl group, or aheteroaryl group substituted with one or two groups chosen from loweralkyl, lower alkyloxy, fluoro, chloro and trifluoromethyl groups.

[0034] In another preferred embodiment, X³ is NH and R⁴ is selected fromR⁶R⁷N(CO), R⁸(CH₂)_(q)CO and R⁸(CH₂)_(q)SO₂.

[0035] Particularly preferred compounds within the invention include:

[0036](2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine2-carbonitrile,

[0037](2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrile,

[0038](2S)-1-[(2′S)-2′-Amino-4′-(pyrazinyl-2″-carbonylamino)butanoyl]pyrrolidine-2-carbonitrile,

[0039](4R)-3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile,

[0040](2S)-1-[N^(ω)-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile,

[0041] 1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine,

[0042](2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile,

[0043] 3-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine,

[0044] 1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine,and

[0045](2S)-1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine2-carbonitrile

[0046] 3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine,

[0047] 3-[N^(ω)-(2-Quinoxaloyl)-L-lysinyl]thiazolidine,

[0048] 3-[N^(ω)-(2-Quinoxaloyl)-L-ornithinyl]thiazolidine,

[0049](2S)-1-[N^(ω)-(2-Quinoxaloyl)-L-ornithinyl]pyrrolidine-2-carbonitrile,

[0050]3-[N^(ω)-(6-Methylpyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine,

[0051] 3-[N^(ω)-(Isoquinoline-3-carbonyl)-L-ornithinyl]thiazolidine,

[0052] 3-[N^(ω)-(6-Trifluoromethylnicotinoyl)-L-ornithinyl]thiazolidine,

[0053](2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrile,

[0054](2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile,

[0055] 3-[N^(ω)-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidine,

[0056] 3-[O-(3-Chlorobenzylcarbamoyl)serinyl]thiazolidine, and

[0057]3-[(2′S)-2′-Amino-5′-oxo-5′-(1″,2″,3″,4″-tetrahydroisoquinolin-2″-yl)pentanoyl]thiazolidine.

[0058] In a second aspect, the present invention comprises apharmaceutical composition for human therapeutic use. The composition ischaracterised in that it has, as an active agent, at least one of thecompounds described above. Such a composition is useful in the treatmentof human diseases. The composition will generally include one or moreadditional components selected from pharmaceutically acceptableexcipients and pharmaceutically active agents other than those of thepresent invention.

[0059] The composition may be presented as a solid or liquidformulation, depending on the intended route of administration. Examplesof solid formulations include pills, tablets, capsules and powders fororal administration, suppositories for rectal or vaginal administration,powders for nasal or pulmonary administration, and patches fortransdermal or transmucosal (such as buccal) administration. Examples ofliquid formulations include solutions and suspensions for intravenous,subcutaneous or intramuscular injection and oral, nasal or pulmonaryadministration. A particularly preferred presentation is a tablet fororal administration. Another preferred presentation, particularly foremergency and critical care, is a sterile solution for intravenousinjection.

[0060] The composition comprises at least one compound according to thepreceding description. The composition may contain more than one suchcompound, but in general it is preferred that it should comprise onlyone. The amount of the compound used in the composition will be suchthat the total daily dose of the active agent can be administered in oneto four convenient dose units. For example, the composition can be atablet containing an amount of compound equal to the total daily dosenecessary, said tablet to be taken once per day. Alternatively, thetablet can contain half (or one third, or one quarter) of the dailydose, to be taken twice (or three or four times) per day. Such a tabletcan also be scored to facilitate divided dosing, so that, for example, atablet comprising a full daily dose can be broken into half andadministered in two portions. Preferably, a tablet or other unit dosageform will contain between 0.1 mg and 1 g of active compound. Morepreferably, it will contain between 1 mg and 250 mg.

[0061] The composition will generally include one or more excipientsselected from those that are recognised as being pharmaceuticallyacceptable. Suitable excipients include, but are not limited to, bulkingagents, binding agents, diluents, solvents, preservatives and flavouringagents. Agents that modify the release characteristics of thecomposition, such as polymers that selectively dissolve in the intestine(“enteric coatings”) are also considered in the context of the presentinvention, to be suitable excipients.

[0062] The composition may comprise, in addition to the compound of theinvention, a second pharmaceutically active agent. For example, thecomposition may include an anti-diabetic agent, a growth-promotingagent, an anti-inflammatory agent or an antiviral agent. However, it isgenerally preferred that the composition comprise only one active agent.

[0063] In a third aspect, the invention comprises a use for thecompounds and compositions described above for the treatment of humandiseases. This aspect can equally be considered to comprise a method oftreatment for such diseases. The diseases susceptible to treatment arethose wherein an inhibition of DP-IV or CD26 results in a clinicalbenefit either directly or indirectly. Direct effects include theblockade of T lymphocyte activation. Indirect effects include thepotentiation of peptide hormone activity by preventing the degradationof these hormones. Examples of diseases include, but are not limited to,auto-immune and inflammatory diseases such as inflammatory bowel diseaseand rheumatoid arthritis, growth hormone deficiency leading to shortstature, polycystic ovary syndrome, impaired glucose tolerance and type2 diabetes. Particularly preferred is the use of the compounds andcompositions for the treatment of impaired glucose tolerance and type 2diabetes, and equally a method of treatment of these diseases by theadministration of an effective amount of a compound or composition aspreviously described.

[0064] The precise details of the treatment, including the dosingregimen, will be established by the attending physician taking intoaccount the general profile of the patient and the severity of thedisease. For diseases such as inflammatory bowel disease that have acutephases of active disease separated by quiescent periods, the physicianmay select a relatively high dose during the acute phase and a lowermaintenance dose for the quiescent period. For chronic diseases such astype 2 diabetes and impaired glucose tolerance, the dosing may need tobe maintained at the same level for an extended period. A dosingschedule of one to four tablets per day, each comprising between 0.1 mgand 1 g (and preferably between 1 mg and 250 mg) of active compoundmight be typical in such a case.

[0065] The compounds according to the invention can be prepared bymethods known in the art. The route chosen will depend on the particularnature of the substituents present in the target molecule. In thefollowing general description the synthetic route is outlined forcompounds wherein m is 1. Compounds with m=2 or 3 can generally beprepared by analogous routes.

[0066] The starting material will usually be an α,ω-diamino acidderivative 5 or an amino dicarboxylic acid derivative 6.

[0067] PG¹ and PG² are “orthogonal” protecting groups—groups that maskthe reactivity of the amine groups and that can each be selectivelyremoved in the presence of the other. Suitable groups are well known inthe literature. PG³ and PG⁴ are protecting groups for carboxylic acids.They are chosen such that they are orthogonal to each other and to theamino protecting groups. Suitable possibilities for PG³ and PG⁴ are alsowell known in the literature. Derivatives of diamino acids according togeneral formula 5 and derivatives of amino dicarboxylic acids accordingto general formula 6 are either items of commerce, or can be preparedfollowing methods described in the literature. In practice, anddepending on the strategy chosen, the starting material will have onlytwo of the three protecting groups present. Either PG³ will be absent toallow the pyrrolidine (or thiazolidine or oxazolidine) residue to beintroduced, or PG¹ or PG⁴ will be absent to allow the side chain to beelaborated.

[0068] Scheme A illustrates the introduction of the pyrrolidine (orthiazolidine or oxazolidine) group as the first step in the preparationof the compounds of the invention.

[0069] Compounds 5^(A) and 6^(A) correspond to 5 and 6 with PG³ as ahydrogen atom (i.e. without the protecting group). The free carboxylicacid can be reacted with a pyrrolidine derivative 7 to give the amide 8or 9. Reaction conditions for achieving this transformation are wellknown in the literature. Suitable reagents include carbodiimides,phosphorus reagents and alkyl chloroformates, and the reaction isusually catalysed by a tertiary amine such as triethylamine ordimethylaminopyridine.

[0070] The reaction depicted in Scheme A is available for allcombinations of R¹ and X¹. However, for the case where R¹ is a nitrilegroup, or where X¹ is a sulphinyl or sulphonyl group, it may beadvantageous to modify the strategy as depicted in Schemes B and C.

[0071] In Scheme B, the R¹ group is introduced as a primary amide andsubsequently transformed into a nitrile by the action of a dehydratingagent such as trifluoroacetic anhydride. In Scheme C, the X¹ group isintroduced as a thioether and subsequently transformed into a sulphoxide(a=1) or sulphone (a=2) by the action of an oxidant such as sodiumperiodate. The modification to the strategy afforded by Scheme C is notpossible if X² is a sulphur atom.

[0072] In Scheme D, compound 5^(D) is the diamino acid derivative 5where the ω-protecting group is a hydrogen atom. The free amine groupreacts readily with sulphonyl chlorides, acyl chlorides and thioacylchlorides, usually in the presence of a tertiary amine, to producesulphonamides 14, amides 15 and thioamides 16 respectively. The reagentsare generally either available per se, or can be prepared from thecorresponding acids. The reaction of scheme D is generally applicable toall the variations of the group R⁸(CH₂)_(q), with the proviso thatcertain of the substituents contemplated for the phenyl and heteroarylrings which are options for R⁸ may require protection. Such substituentsand the appropriate protection will generally be obvious to thosefamiliar with the art.

[0073] Reagents: i) COCl₂; ii) CSCl₂; iii) R⁸(CH₂)_(q)OH; iv) R⁶R⁷NH; v)R⁸(CH₂)_(q)OCOCl; vi) R⁸(CH₂)_(q)OCSCl; vii) R⁶R⁷NCOCl; viii) R⁶R⁷NCSCl.

[0074] Scheme E illustrates the elaboration of 5^(D) to give carbamatesand ureas, and their thio analogues. When R⁵ is other than a hydrogenatom, compound 5^(D) can be converted to the corresponding carbamoylchloride 17 or thiocarbamoyl chloride 18 by reaction with phosgene orthiophosgene. Other reagents are known in the art to be functionallyequivalent to these toxic reagents and they may also be used. When R⁵ ishydrogen, the intermediate formed is an isocyanate or isothiocyanate,but this behaves functionally as an equivalent of the correspondingchloride. Intermediates 17 and 18 are not normally isolated, but aretreated directly with alcohols to give carbamates 19 and thiocarbamates20. Treatment of these same intermediates with amines leads to theformation of ureas 21 and thioureas 22. Alternatively, 5^(D) may bereacted directly with a chloroformate or chlorothioformate to give thecarbamate or thiocarbamate, or, when neither R⁶ nor R⁷ is hydrogen, witha chloroformamide or chlorothioformamide to give the urea or thiourea.When R⁶ or R⁷ is a hydrogen atom the chloroformamide orchlorothioformamide will tend to be unstable, in which case theisocyanate (eg. R⁶—NCO) or isothiocyanate (e.g. R⁶—NCS) is used. Asdiscussed previously for Scheme D, certain substituents within theembodiments of R⁸ may require appropriate protection.

[0075] Scheme F illustrates the elaboration of the side chain of theamino dicarboxylic acid series. The ω-deprotected acid 6^(F) can bereacted under a variety of conditions with an amine to give amide 23.The condensation may be promoted by a dehydrating reagent such as acarbodiimide or a phosphorus reagent. Alternatively the acid may beconverted into a more reactive derivative, such as by treatment withoxalyl chloride or thionyl chloride to give the corresponding acidchloride, which will react directly with an amine. The thioamide 24 maybe obtained by treating the amide 23 with Lawesson's reagent.

[0076] When all the groups have been elaborated the final protectinggroup is removed and the product is isolated and purified using standardtechniques.

[0077] These general methods are further illustrated in the following,non limiting examples.

EXAMPLES

[0078] Abbreviations

[0079] The following abbreviations have been used. DMFN,N-Dimethyformamide h Hour(s) hplc High pressure liquid chromatographymin Minute(s) pet. ether Petroleum ether fraction boiling at 60-80° C.PyBOP ® (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate PyBroP ® Bromotripyrrolidinophosphoniumhexafluorophosphate TFA Trifluoroacetic acid

Example 1(2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0080]

[0081] A. N-(2-Nitrobenzenesulphenyl)-L-proline

[0082] L-Proline (25 g, 217 mmol) was dissolved in 2M NaOH (110 mL, 220mmol) and dioxan (120 mL). A solution of 2-nitrobenzenesulphenylchloride (42 g, 222 mmol) in dioxan (60 mL) was slowly added at the sametime as 2M NaOH (110 mL, 220 mmol). After 2 h at room temperature thereaction mixture was poured into water (500 mL) and the solid filteredoff. The pH of the filtrate was adjusted to pH3 with 2M HCl and thesolution was extracted with ethyl acetate (3×500 mL). The combinedorganic extracts were washed with water (4×200 mL) and brine (1×200 mL),dried (Na₂SO₄) and evaporated in vacuo to give an orange solididentified as N-(2-nitrobenzenesulphenyl)-L-proline (58.1 g, 217 mmol,100%).

[0083] B. N-(2-Nitrobenzenesulphenyl)-L-proline succinimidyl Ester

[0084] N-(2-Nitrobenzenesulphenyl)-L-proline (57.9 g, 216 mmol) wasdissolved in CH₂Cl₂/DMF (9:1, 500 mL). N-Hydroxysuccinimide (37.3 g, 324mmol) and water-soluble carbodiimide (51.8 g, 260 mmol) were added.After 18 h at room temperature the solvent was removed in vacuo and theresidue was taken up in ethyl acetate (1000 mL). The solution was washedwith water (4×200 mL) and brine (1×200 mL), dried (Na₂SO₄) andevaporated in vacuo to give a yellow solid identified asN-(2-nitrobenzenesulphenyl)-L-proline succinimidyl ester (78.9 g, 216mmol, 100%).

[0085] C. N-(2-Nitrobenzenesulphenyl)-L-prolinamide

[0086] N-(2-Nitrobenzenesulphenyl)-L-proline succinimidyl ester (78.5 g,215 mmol) was dissolved in dioxan (500 mL). Ammonia (35%, 100 mL) wasadded, After stirring at room temperature for 2 h the reaction mixturewas poured into water (700 mL). The precipitate was filtered off, washedwith water (200 mL), dried over P₂O₅ and recrystallised from ethylacetate/pet ether to give a yellow solid identified asN-(2-nitrobenzenesulphenyl)-L-prolinamide (49.6 g, 185 mmol, 86%).

[0087] D. (2S)-N-(2-Nitrobenzenesulphenyl)pyrrolidine-2-carbonitrile

[0088] N-(2-Nitrobenzenesulphenyl)-L-prolinamide (49 g, 183 mmol) wasdissolved in dry THF(300 mL). The solution was cooled to 0° C.,triethylamine (36.7 g, 367 mmol) was added followed by the slow additionof trifluoroacetic anhydride (77 g, 367 mmol). The pH was adjusted topH9 with triethylamine. After 30 min the reaction mixture was dilutedwith ethyl acetate (500 mL), washed with water (1×200 mL) and brine(1×200 mL), dried (Na₂SO₄) and evaporated in vacuo to give an orange oilwhich was purified by flash chromatography (eluant: 80% pet ether, 20%ethyl acetate) to give a yellow solid identified as(2S)-N-(2-nitrobenzenesulphenyl)pyrrolidine-2-carbonitrile (38.9 g, 150mmol, 82%).

[0089] E. (2S)-Pyrrolidine-2-carbonitrile Hydrochloride

[0090] (2S)-N-(2-Nitrobenzenesulphenyl)pyrrolidine-2-carbonitrile (38.5g, 149 mmol) was dissolved in diethyl ether (200 mL). 4M HCl/Dioxan (150mL, 600 mmol) was slowly added. After 2 h at room temperature thereaction mixture was poured into diethyl ether (1000 mL). The solid wasfiltered off, washed with diethyl ether (500 mL) and recrystallised frommethanol/diethyl ether to give a white solid identified as(2S)-pyrrolidine-2-carbonitrile hydrochloride (18.9 g, 142.5 mmol, 96%).

[0091] F.(2S)-1-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]-pyrrolidine-2-carbonitrile.

[0092]N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithine(2.5 g, 7.4 mmol) was dissolved in CH₂Cl₂ (50 mL). This solution wascooled to 0° C., (2S)-pyrrolidine-2-carbonitrile hydrochloride (1.2 g,9.1 mmol) and PyBOP® (4.3 g, 8.23 mmol) were added, and the pH adjustedto pH9 with triethylamine. After 18 h at 0° C. to room temperature thesolvent was removed in vacuo and the residue was taken up in ethylacetate (200 mL). This solution was washed with 0.3M KHSO₄ (2×50 mL),sat. NaHCO₃ (2×50 mL), water (2×50 mL) and brine (1×50 mL), dried(Na₂SO₄) and evaporated in vacuo to give a yellow oil. This was purifiedby flash chromatography (eluant: 80% ethyl acetate, 20% pet. ether) togive a colourless oil identified as(2S)-1-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile(2.98 g, 7.16 mmol, 97%).

[0093] G.(2S)-1-[N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0094](2S)-1-[N^(α)-tert-Butyloxycarbonyl-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile(2.8 g, 6.7 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1h at room temperature the solvent was removed in vacuo. The residue waspurified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrileinto 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing theproduct were lyophilised to give a colourless oil identified as(2S)-1-[N^(ω)(-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitriletrifluoroacetate (1.5 g, 3.48 mmol, 52%).

[0095] [M+H]⁺=317.3

Example 2(2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0096]

[0097] A.(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-L-prolinamide

[0098]N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysine(5 g, 10.7 mmol) was dissolved in CH₂Cl₂ (100 mL). The solution wascooled to 0° C., L-prolinamide (1.78 g, 11.7 mmol) and PyBOP® (6.7 g,12.8 mmol) were added, and the pH adjusted to pH9 with triethylamine.After 18 h at 0° C. to room temperature the solvent was removed in vacuoand the residue was taken up in ethyl acetate (200 mL). The solution waswashed with 0.3M KHSO₄ (2×50 mL), sat. NaHCO₃ (2×50 mL), water (2×50 mL)and brine (1×50 mL), dried (Na₂SO₄) and evaporated in vacuo. The residuewas purified by flash chromatography (eluant: 2% methanol, 98%chloroform) to give a colourless oil identified as(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-L-prolinamide(4.05 g, 7.2 mmol, 67%).

[0099] B.(2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-pyrrolidine-2-carbonitrile

[0100](N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-L-prolinamide(3.95 g, 7.02 mmol) was dissolved in dry THF (100 mL). The solution wascooled to 0° C., triethylamine (1.4 g, 14 mmol) was added followed bythe slow addition of trifluoroacetic anhydride (2.97 g, 14.1 mmol). ThepH was adjusted to pH9 with triethylamine. After 30 min the reactionmixture was diluted with ethyl acetate (100 mL), washed with water (1×50mL) and brine (1×50 mL), dried (Na₂SO₄) and evaporated in vacuo to givean orange oil. The residue was purified by flash chromatography (eluant:60% pet ether, 40% ethyl acetate) to give a colourless oil identified as(2S)-1-(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile(3.3 g, 6.11 mmol, 87%).

[0101] C.(2S)-1-(N^(α)(-tert-Butyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile

[0102](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile(3.1 g, 5.7 mmol) was dissolved in THF (80 mL). Diethylamine (20 mL) wasadded. After 2 h at room temperature the solvent was removed in vacuo.The residue was purified by flash chromatography (eluant: 90%chloroform, 7% methanol, 3% triethylamine) to give a colourless oilidentified as(2S)-1-(N^(α)-(tert-butyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile(1.63 g, 5.03 mmol, 89%).

[0103] D.(2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile

[0104](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile(100 mg, 0.31 mmol) was dissolved in CH₂Cl₂/DMF (9:1, 20 mL). To thissolution at 0° C. was added 1-hydroxybenzotriazole hydrate (84 mg, 0.62mmol), water-soluble carbodiimide (76 mg, 0.38 mmol),2-pyrazinecarboxylic acid (43 mg, 0.35 mmol) and triethylamine (65 mg,0.65 mmol). After 18 h at 0° C. to room temperature the solvent wasremoved in vacuo and the residue was taken up in ethyl acetate (70 mL).This solution was washed with 0.3M KHSO₄ (2×20 mL), sat. NaHCO₃ (2×20mL), water (2×20 mL) and brine (1×20 mL), dried (Na₂SO₄) and evaporatedin vacuo to give a yellow oil. The residue was purified by flashchromatography (eluant: 2% methanol, 98% chloroform) to give acolourless oil identified as(2S)-1-(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile(124 mg, 0.29 mmol, 93%).

[0105] E.(2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0106](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile(110 mg, 0.26 mmol) was dissolved in trifluoroacetic acid (5 mL). After1 h at room temperature the solvent was removed in vacuo. The residuewas purified by preparative hplc (Vydac C18, 5 to 50% 0.1%TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractionscontaining the product were lyophilised to give a colourless oilidentified as(2S)-1-[N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitriletrifluoroacetate (66 mg).

[0107] [M+H]⁺=331.1

Example 3(4R)-3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrileTrifluoroacetate

[0108]

[0109] A. (4R)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxamide

[0110] (4R)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxylic acid(12.5 g,54.1 mmol) was dissolved in CH₂Cl₂/DMF (9:1, 150 mL). To thissolution at 0° C. was added 1-hydroxybenzotriazole hydrate (14.6 g, 108mmol) and water-soluble carbodiimide (13.0 g, 65 mmol). After 1 h at 0°C. ammonia (35%, 50 mL) was added. After 18 h at 0° C. to roomtemperature the solvent was removed in vacuo and the residue was takenup in ethyl acetate (500 mL). The solution was washed with 0.3M KHSO₄(2×100 mL), sat. NaHCO₃ (2×100 mL), water (2×100 mL) and brine (1×100mL), dried (Na₂SO₄) and evaporated in vacuo to give a yellow oil. Theresidue was purified by flash chromatography (eluant: 2% methanol, 98%chloroform) to give a colourless oil identified as(4R)-3-(tert-butyloxycarbonyl)thiazolidine-4-carboxamide (8.9 g, 38.4mmol, 71%).

[0111] B. (4R)-Thiazolidine4-carboxamide Hydrochloride

[0112] (4S)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxamide (8.6 g,37.1 mmol) was dissolved in 4M HCl/dioxan (50 mL). After 1 h at roomtemperature the solvent was evaporated in vacuo to give a white solididentified as (4R)-thiazolidine-4-carboxamide hydrochloride (6.2 g, 36.8mmol, 99%).

[0113] C.(4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]-thiazolidine4-carboxamide

[0114]N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysine(5 g, 10.7 mmol) was dissolved in CH₂Cl₂ (100 mL). This solution wascooled to 0° C., (4R)-thiazolidine-4-carboxamide hydrochloride (1.78 g,11.7 mmol) and PyBOP® (6.7 g, 12.8 mmol) were added, and the pH wasadjusted to pH9 with triethylamine. After 18 h at 0° C. to roomtemperature the solvent was removed in vacuo and the residue was takenup in ethyl acetate (200 mL). The solution was washed with 0.3M KHSO₄(2×50 mL), sat. NaHCO₃ (2×50 mL), water (2×50 mL) and brine (1×50 mL),dried (Na₂SO₄) and evaporated in vacuo to give a yellow oil. The residuewas purified by flash chromatography (eluant: 2% methanol, 98%chloroform) to give a colourless oil identified as(4R)-3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carboxamide(2.81 g, 4.8 mmol, 44%).

[0115] D.(4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]-thiazolidine-4-carbonitrile

[0116](4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carboxamide(2.7 g, 4.7 mmol) was dissolved in dry THF (100 mL). The solution wascooled to 0° C., triethylamine (1.0 g, 10 mmol) was added followed bythe slow addition of trifluoroacetic anhydride (2.0 g, 9.5 mmol). The pHwas adjusted to pH9 with triethylamine. After 30 min the reactionmixture was diluted with ethyl acetate (100 mL), washed with water (1×50mL) and brine (1×50 mL), dried (Na₂SO₄) and evaporated in vacuo. Theresidue was purified by flash chromatography (eluant: 60% pet ether, 40%ethyl acetate) to give a colourless oil identified as(4R)-3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile(2.14 g, 3.81 mmol, 82%).

[0117] E.(4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-L-lysinyl]thiazolidine4-carbonitrile

[0118](4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile(1.9 g, 3.4 mmol) was dissolved in THF (40 mL). Diethylamine (10 mL) wasadded. After 2 h at room temperature the solvent was removed in vacuo.The residue was purified by flash chromatography (eluant: 90%chloroform, 7% methanol, 3% triethylamine) to give a colourless oilidentified as(4R)-3-[N^(α)-(tert-butyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile(863 mg, 2.5 mmol, 75%).

[0119] F.(4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile

[0120](4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile(100 mg, 0.29 mmol) was dissolved in CH₂Cl₂ (20 mL). To this solution at0° C. 2-pyrazinecarboxylic acid (43 mg, 0.35 mmol) and PyBOP® (170 mg,0.33 mmol) were added and the pH was adjusted to pH9 with triethylamine.After 18 h at 0° C. to room temperature the solvent was removed in vacuoand the residue was taken up in ethyl acetate (70 mL). The solution waswashed with 0.3M KHSO₄ (2×20 mL), sat. NaHCO₃ (2×20 mL), water (2×20 mL)and brine (1×20 mL), dried (Na₂SO₄) and evaporated in vacuo. The residuewas purified by flash chromatography (eluant: 2% methanol, 98%chloroform) to give a colourless oil identified as(4R)-3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile(112 mg, 0.25 mmol, 86%).

[0121] G.(4R)-3-[N^(ω)-Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrileTrifluoroacetate

[0122](4R)-3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile(110 mg, 0.26 mmol) was dissolved in trifluoroacetic acid (5 mL). After1 h at room temperature the solvent was removed in vacuo. The residuewas purified by preparative hplc (Vydac C18, 5 to 50% 0.1%TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractionscontaining the product were lyophilised to give a colourless oilidentified as(4R)-3-[N^(ω)-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitriletrifluoroacetate (57 mg).

[0123] [M+H]⁺=349.1

Example 4(2S)-1-[N^(ω)-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrileDihydrochloride

[0124]

[0125] A.(2S)-1-[N-(tert-Butyloxycarbonyl)-O^(ω)-methyl-L-glutamyl]pyrrolidine-2-carbonitrile

[0126] N-(tert-Butyloxycarbonyl)-O^(ω)-methyl-L-glutamic acid (1.0 g,3.83 mmol) was dissolved in CH₂Cl₂/DMF (9:1, 20 mL). To this solution at0° C. were added 1-hydroxybenzotriazole hydrate (788 mg, 5.84 mmol),water-soluble carbodiimide (877 mg, 4.38 mmol),(2S)-pyrrolidine-2-carbonitrile hydrochloride (609 mg, 4.6 mmol) andtriethylamine (65 mg, 0.65 mmol). After 18 h at 0° C. to roomtemperature the solvent was removed in vacuo and the residue was takenup in ethyl acetate (70 mL), this solution was washed with 0.3M KHSO₄(2×20 mL), sat. NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20 mL(Na₂SO₄) and evaporated in vacuo. The residue was purified by flashchromatography (eluant: 50% ethyl acetate, 50% pet. ether) to give abrown oil identified as(2S)-1-[N-(tert-butyloxycarbonyl)-O^(ω)-methyl-L-glutamyl]pyrrolidine-2-carbonitrile(290 mg, 0.86 mmol, 22%).

[0127] B.(2S)-1-[N-(tert-Butyloxycarbonyl)-L-glutamyl]pyrrolidine-2-carbonitrile

[0128](2S)-1-[N-(tert-Butyloxycarbonyl)-O^(ω)-methyl-L-glutamyl]pyrrolidine-2-carbonitrile(250 mg, 0.74 mmol) was dissolved in dioxan (5 mL). 1M Lithium hydroxide(1.1 mL, 1.1 mmol) was added. After 1 h at room temperature the solventwas removed in vacuo and the residue was taken up in ethyl acetate (70mL). This solution was washed with 1M KHSO₄ (2×20 mL), water (2×20 mL)and brine (1×20 mL), dried (Na₂SO₄) and evaporated in vacuo to give acolourless oil identified as(2S)-1-[N-(tert-butyloxycarbonyl)-L-glutamy]pyrrolidine-2-carbonitrile(200 mg, 0.61 mmol, 83%).

[0129] C.(2S)-1-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile

[0130](2S)-1-[N-(tert-Butyloxycarbonyl)-L-glutamyl]pyrrolidine-2-carbonitrile(30 mg, 0.093 mmol) was dissolved in CH₂Cl₂/DMF (9:1, 10 mL). To thissolution at 0° C. was added 1-hydroxybenzotriazole hydrate (21 mg, 0.16mmol), water-soluble carbodiimide (21 mg, 0.105 mmol),3-(aminomethyl)pyridine (11 mg, 0.1 mmol) and triethylamine(20 mg, 0.2mmol). After 18 h at 0° C. to room temperature the solvent was removedin vacuo and the residue was taken up in ethyl acetate (70 mL). Thesolution was washed with 0.3M KHSO₄ (2×20 mL), sat. NaHCO₃ (2×20 mL),water (2×20 mL) and brine (1×20 mL), dried (Na₂SO₄) and evaporated invacuo to give a yellow oil. The residue was purified by flashchromatography (eluant: 5% methano, 97% chloroform) to give a colourlessoil identified as(2S)-1-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile(16.6 mg, 0.04 mmol, 44%).

[0131] D.(2S)-1-[N^(ω)-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrileDihydrochloride

[0132](2S)-1-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidin-2-carbonitrile(17 mg, 0.04 mmol) was dissolved in 4M HCl/dioxan (5 mL). After 1 h atroom temperature the solvent was removed in vacuo to give a white solididentified as(2S)-1-[N^(ω)-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitriledihydrochloride (17 mg, 0.04 mmol, 100%).

[0133] [M+H]⁺=316.2

Example 5 1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidineTrifluoroacetate

[0134]

[0135] A.1-[N^(ω)-(Benzyloxycarbonyl)-N^(α)-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine

[0136]N^(ω)-(Benzyloxycarbonyl)-N^(α)-(tert-butyloxycarbonyl)-L-ornithine(5.49 g, 15 mmol) was dissolved in CH₂Cl₂/DMF (9:1, 100 mL). To thissolution at 0° C. was added 1-hydroxybenzotriazole hydrate (3.37 g, 22mmol), water-soluble carbodiimide (3.46 g, 18 mmol), pyrrolidine (1.28g, 18 mmol) and triethylamine (200 mg, 20 mmol). After 18 h at 0° C. toroom temperature the solvent was removed in vacuo and the residue wastaken up in ethyl acetate (200 mL). The solution was washed with 0.3MKHSO₄ (2×50 mL), sat. NaHCO₃ (2×50 mL), water (2×50 mL) and brine (1×50mL), dried (Na₂SO₄) and evaporated in vacuo. The residue was purified byflash chromatography (eluant: 90% ethyl acetate, 10% pet. ether) to givea colourless oil identified as1-[N^(ω)-(benzyloxycarbonyl)-N^(α)-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine(5.15 g, 12.3 mmol, 82%).

[0137] B. 1-[N^(α)-(tert-Butyloxycarbonyl)-L-ornithinyl]pyrrolidine

[0138]1-[N^(ω)-(1-(Benzyloxycarbonyl)-N^(α)-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine(2.15 g, 5.13 mmol) was dissolved in methanol (80 mL). This solution washydrogenated over 10% Pd/C (400 mg). After 2 h the catalyst was filteredoff and washed with methanol (50 mL). The combined filtrates wereevaporated in vacuo to give an off white solid identified as1-[N^(α)-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine (1.35 g, 4.74mmol, 94%).

[0139] C.1-[N^(α)-tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine

[0140] 1-[N^(α)-(tert-Butyloxycarbonyl)-L-ornithinyl]pyrrolidine (100mg, 0.35 mmol) was dissolved in CH₂Cl₂ (20 mL). To this solution at 0°C. were added PyBroP® (195 mg, 0.4 mmol), 2-pyrazinecarboxylic acid (50mg, 0.4 mmol) and triethylamine (100 mg, 1.0 mmol). After 18 h at 0° C.to room temperature the solvent was removed in vacuo and the residue wastaken up in ethyl acetate (70 mL). The solution was washed with 0.3MKHSO₄ (2×20 mL), sat. NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20mL), dried (Na₂SO₄) and evaporated in vacuo. The residue was purified byflash chromatography (eluant: 3% methanol, 97% chloroform) to give asticky white solid identified as1-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine(90 mg, 0.25 mmol, 66%).

[0141] D. 1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidineTrifluoroacetate

[0142]1-[N^(α)-(ert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine(90 mg, 0.23 mmol) was dissolved in 4M HCl/dioxan (15 mL). After 45 minat room temperature the solvent was removed in vacuo. The residue waspurified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrileinto 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing theproduct were lyophilised to give a colourless oil identified as1-[N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidinetrifluoroacetate (51 mg).

[0143] [M+H]⁺=292.1

Example 6 3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidinetrifluoroacetate

[0144]

[0145] A.3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-ornithinyl]thiazolidine

[0146]N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-ornithine(2.73 g, 6 mmol) was dissolved in CH₂Cl₂/DMF (9:1, 100 mL). To thissolution at 0° C. were added 1-hydroxybenzotriazole hydrate (1.53 g, 10mmol), water-soluble carbodiimide (1.34 g, 7 mmol), thiazolidine (1.28g, 18 mmol) and triethylamine (80 mg, 8 mmol). After 18 h at 0° C. toroom temperature the solvent was removed in vacuo and the residue wastaken up in ethyl acetate (100 mL). The solution was washed with 0.3MKHSO₄ (2×25 mL), sat. NaHCO₃ (2×25 mL), water (2×25 mL) and brine (1×25mL), dried (Na₂SO₄) and evaporated in vacuo. The residue was purified byflash chromatography (eluant: 75% ethyl acetate, 25% pet. ether) to givea white solid identified as3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-ornithinyl]thiazolidine(2.55 g, 4.85 mmol, 81%).

[0147] B. 3-[N^(α)-(tert-Butyloxycarbonyl)-L-ornithinyl]thiazolidine

[0148]3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(9-fluorenylmethyloxycarbonyl)-L-ornithinyl]thiazolidine(1.15 g, 2.13 mmol) was dissolved in acetonitrile (20 mL). Diethylamine(5 mL) was added. After 90 min at room temperature the solvent wasremoved in vacuo and the residue was purified by flash chromatography(eluant: 90% chloroform, 7% methanol, 3% triethylamine) to give a paleyellow oil identified as3-[N^(α)-(tert-butyloxycarbonyl)-L-ornithinyl]thiazolidine (530 mg, 1.67mmol, 78%).

[0149] C.3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine

[0150] 3-[N^(α)-(tert-Butyloxycarbonyl)-L-ornithinyl]thiazolidine (80mg, 0.27 mmol) was dissolved in CH₂Cl₂ (20 mL). To this solution at 0°C. were added PyBroP® (146 mg, 0.3 mmol), 2-pyrazinecarboxylic acid (37mg, 0.3 mmol) and triethylamine (90 mg, 0.9 mmol). After 18 h at 0° C.to room temperature the solvent was removed in vacuo and the residue wastaken up in ethyl acetate (70 mL). The solution was washed with 0.3MKHSO₄ (2×20 mL), sat. NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20mL), dried (Na₂SO₄) and evaporated in vacuo. The residue was purified byflash chromatography (eluant: 3% methanol, 97% chloroform) to give asticky white solid identified as3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine(45 mg, 0.11 mmol, 41%).

[0151] D. 3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidineTrifluoroacetate

[0152]3-[N^(α)-(-(tert-Butyloxycarbonyl)-N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine(45 mg, 0.11 mmol) was dissolved in 4M HCl/dioxan (10 mL). After 45 minat room temperature the solvent was removed in vacuo. The residue waspurified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrileinto 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing theproduct were lyophilised to give a colourless oil identified as3-[N^(ω)-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidinetrifluoroacetate (14 mg).

[0153] [M+H]⁺=310.0

Example 7(2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0154]

[0155] A.(2S)-1-[S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0156] S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteine (660mg, 2.26 mmol) was dissolved in CH₂Cl₂ (30 mL). To this solution at 0°C. were added (2S)-pyrrolidine-2-carbonitrile hydrochloride (250 mg,1.89 mmol) and PyBOP® (1.3 g, 2.49 mmol), and the pH adjusted to pH9with triethylamine. After 18 h at 0° C. to room temperature the solventwas removed in vacuo and the residue was taken up in ethyl acetate (150mL). The solution was washed with 0.3M KHSO₄ (2×30 mL), sat. NaHCO₃(2×30 mL), water (2×30 mL) and brine (1×30 mL), dried (Na₂SO₄) andevaporated in vacuo. The residue was purified by flash chromatography(eluant: 75% ethyl acetate, 25% pet. ether) to give a colourless oilidentified as(2S)-1-[S-(acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]-pyrrolidine-2-carbonitrile(650 mg, 1.76 mmol, 78%).

[0157] B.(2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0158](2S)-1-[S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile(610 mg, 1.65 mmol) was dissolved in trifluoroacetic acid (30 mL). After1 h at room temperature the solvent was removed in vacuo to give acolourless oil identified as(2S)-1-[S-(acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitriletrifluoroacetate (620 mg, 1.61 mmol, 98%).

[0159] [M+H]⁺=271.0

Example 8(2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0160]

[0161] A.(2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-(tert-butyloxycarbonylamino)-3′-methylbutanoyl]pyrrolidine-2-carbonitrile

[0162] S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)penicillamine (720mg, 2.25 mmol) was dissolved in CH₂Cl₂ (30 mL). To this solution at 0°C. were added (2S)-pyrrolidine-2-carbonitrile hydrochloride (270 mg,2.04 mmol) and PyBOP® (1.3 g, 2.49 mmol), and the pH adjusted to pH9with triethylamine. After 18 h at 0° C. to room temperature the solventwas removed in vacuo and the residue was taken up in ethyl acetate (150mL). The solution was washed with 0.3M KHSO₄ (2×30 mL), sat. NaHCO₃(2×30 mL), water (2×30 mL) and brine (1×30 mL), dried (Na₂SO₄) andevaporated in vacuo. The residue was purified by flash chromatography(eluant: 75% ethyl acetate, 25% pet. ether) to give a colourless oilidentified as(2S)-1-[(2′R)-3′-(acetylaminomethylthio)-2′-(tert-butyloxycarbonylamino)-3′-methylbutanoyl]pyrrolidine-2-carbonitrile(742 mg, 1.86 mmol, 83%).

[0163] B.(2S)-1-[(2′-R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0164](2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-(tert-butyloxycarbonylamino)-3′-methylbutanoyl]pyrrolidine-2-carbonitrile(710 mg, 1.78 mmol) was dissolved in trifluoroacetic acid (30 mL). After1 h at room temperature the solvent was removed in vacuo to give acolourless oil identified as(2S)-1-[(2′R)-3′-(acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitriletrifluoroacetate (560 mg, 1.36 mmol, 76%).

[0165] [M+H]⁺=299.1

Example 9(2S)-1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0166]

[0167] A.(2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile

[0168](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrilewas prepared by the method described for the lysine derivative inExample 2.

[0169] B.(2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)-pyrrolidine-2-carbonitrile

[0170](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile(80 mg, 0.26 mmol) was dissolved in CH₂Cl₂ (20 mL). To this solution wasadded 2-chloropyridine-3-carbonyl chloride (55 mg, 0.32 mmol) and the pHadjusted to pH9 with triethylamine. After 18 h at room temperature thesolvent was removed in vacuo and the residue was taken up in ethylacetate (70 mL). The solution was washed with 0.3M KHSO₄ (2×20 mL), sat.NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na₂SO₄)and evaporated in vacuo. The residue was purified by flashchromatography (eluant: 95% ethyl acetate, 5% pet. ether) to give acolourless oil identified as(2S)-1-(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile(60 mg, 0.14 mmol, 53%).

[0171] C.(2S)-1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0172](2S)-1-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile(60 mg, 0.14 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1h at room temperature the solvent was removed in vacuo. The residue waspurified by

[0173] [M+H]⁺=299.1

Example 9(2S)-1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0174]

[0175] A.(2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile

[0176](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrilewas prepared by the method described for the lysine derivative inExample 2.

[0177] B.(2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)-pyrrolidine-2-carbonitrile

[0178](2S)-1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile(80 mg, 0.26 mmol) was dissolved in CH₂Cl₂ (20 mL). To this solution wasadded 2-chloropyridine-3-carbonyl chloride (55 mg, 0.32 mmol) and the pHadjusted to pH9 with triethylamine. After 18 h at room temperature thesolvent was removed in vacuo and the residue was taken up in ethylacetate (70 mL). The solution was washed with 0.3M KHSO₄ (2×20 mL), sat.NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na₂SO₄)and evaporated in vacuo. The residue was purified by flashchromatography (eluant: 95% ethyl acetate, 5% pet. ether) to give acolourless oil identified as(2S)-1-(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile(60 mg, 0.14 mmol, 53%).

[0179] C.(2S)-1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0180](2S)-1-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile(60 mg, 0.14 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1h at room temperature the solvent was removed in vacuo. The residue waspurified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrileinto 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing theproduct were lyophilised to give a white solid identified as(2S)-1-[N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitriletrifluoroacetate (52 mg).

[0181] [M+H]⁺=350.1

Example 10 31-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidineHydrochloride

[0182]

[0183] A.1-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine

[0184] 1-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine (20 mg,0.069 mmol) was dissolved in CH₂Cl₂ (5 mL). To this solution was added2-chloropyridine-3-carbonyl chloride (14 mg, 0.076 mmol) and the pHadjusted to pH9 with triethylamine. After 1 h at room temperature thesolvent was removed in vacuo and the residue was taken up in ethylacetate (70 mL). The solution was washed with 0.3M KHSO₄ (2×20 mL), sat.NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na₂SO₄)and evaporated in vacuo. The residue was purified by flashchromatography (eluant: 10% methanol, 90% dichloromethane) to give acolourless oil identified as1-(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine(19 mg, 0.045 mmol, 63%).

[0185] B. 1-[N^(ω)-(2Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidineHydrochloride

[0186]1-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine(19 mg, 0.045 mmol) was dissolved in 4M HCl/dioxan (10 mL). After 45 minat room temperature the solvent was removed in vacuo to give a whitesolid identified as1-[N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidinehydrochloride (15 mg).

[0187] [M+H]⁺=325.1

Example 113-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidineHydrochloride

[0188]

[0189] A.3-(N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)thiazolidine

[0190] 3-(N^(α)-(tert-Butyloxycarbonyl)-L-ornithyl)thiazolidine (136 mg,0.45 mmol) was dissolved in CH₂Cl₂ (10 mL). To this solution was added2-chloropyridine3-carbonyl chloride (88 mg, 0.5 mmol) and the pHadjusted to pH9 with triethylamine. After 1 h at room temperature thesolvent was removed in vacuo and the residue was taken up in ethylacetate (70 mL). The solution was washed with 0.3M KHSO₄ (2×20 mL), sat.NaHCO₃ (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na₂SO₄)and evaporated in vacuo. The residue was purified by flashchromatography (eluant: 1.5% methanol, 98.5% dichloromethane) to give acolourless oil identified as3-(N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithyl)thiazolidine(30 mg, 0.068 mmol, 15%).

[0191] B.3-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidineHydrochloride

[0192]3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine(30 mg, 0.068 mmol) was dissolved in 4M HCl/dioxan (10 mL). After 45 minat room temperature the solvent was removed in vacuo to give a whitesolid identified as1-[N^(ω)-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidinehydrochloride (25 mg).

[0193] [M+H]⁺=342.1

Example 12(2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2carbonitrileTrifluoroacetate

[0194]

[0195] A.S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteine

[0196] N-(tert-Butyloxycarbonyl)-L-cysteine (3.5 g, 15.8 mmol), benzyl2-bromoacetate (3.7 g, 16.1 mmol) and triethylamine (1.8 g, 18.0 mmol)were dissolved in THF (100 mL). After 18 hours at room temperature thereaction mixture was diluted with ethyl acetate (100 mL), washed with0.3M KHSO₄, sat NaHCO₃, water and brine, dried (Na₂SO₄) and evaporated.The residue was purified by flash chromatography (eluant 95% chloroform,4% methanol, 1% acetic acid) yielding a colourless oil identified asS-(benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteine (5.2 g,14.1 mmol, 89%).

[0197] B.(2S)-1-[S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0198] S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteine(5.10 g, 13.8 mmol) was dissolved in CH₂Cl₂ (200 mL). This solution wascooled to 0° C., (2S)-pyrrolidine-2-carbonitrile hydrochloride (2.1 g,15.8 mmol) and PyBOP (8.0 g, 15.3 mmol) were added, and the pH wasadjusted to pH9 with triethylamine. After 18 h at 0° C. to roomtemperature the solvent was removed in vacuo and the residue was takenup in ethyl acetate (150 mL). This solution was washed with 0.3M KHSO₄(1×50 mL), sat. NaHCO₃ (1×50 mL), water (1×50 mL) and brine (1×50 mL),dried (Na₂SO₄) and evaporated in vacuo to give a yellow oil. This waspurified by flash chromatography (eluant: 40% ethyl acetate, 60% pet.ether) to give a colourless oil identified as(2S)-1-[S-(benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile(5.82 g, 13.0 mmol, 94%).

[0199] C.(2S)-1-[N-(tert-Butyloxycarbonyl)-S-(carboxymethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0200](2S)-1-[S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile(1.31 g, 2.9 mmol) was dissolved in THF(100 mL). Aqueous lithiumhydroxide (1M, 3.5 mL, 3.5 mmol) was added. After 3 hours at roomtemperature the reaction mixture was diluted with ethyl acetate (100mL), washed with 1M citric acid, water and brine, dried (Na₂SO₄) andevaporated in vacuo to give a colourless oil. This was purified by flashchromatography (eluant: 97% chloroform, 2% methanol, 1% acetic acid) togive a colourless oil identified as(2S)-1-[N-(tert-butyloxycarbonyl)-S-(carboxymethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile(860 mg, 2.4 mmol, 82%).

[0201] D.(2S)-1[N-(Tert-Butyloxycarbonyl)-S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0202](2S)-1-[N-(tert-Butyloxycarbonyl)-S-(carboxymethyl)-L-cysteinyl]pyrrolidine-2-carbonitrite(150 mg, 0.42 mmol) was dissolved in CH₂Cl₂ (20 mL). This solution wascooled to 0° C., 3-(aminomethyl)pyridine (53 mg, 0.5 mmol) and PyBOP(270 mg, 0.52 mmol) were added, and the pH adjusted to pH9 withtriethylamine. After 18 h at 0° C. to room temperature the solvent wasremoved in vacuo and the residue was taken up in ethyl acetate (70 mL).This solution was washed with 0.3M KHSO₄ (1×20 mL), sat. NaHCO₃ (1×20mL), water (1×20 mL) and brine (1×20 mL), dried (Na₂SO₄) and evaporatedin vacuo to give a yellow oil. This was purified by flash chromatography(eluant: 96% chloroform, 4% methanol) to give a colourless oilidentified as(2S)-1-[N-(tert-butyloxycarbonyl)-S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile(170 mg, 0.38 mmol, 91%).

[0203] E.(2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrileTrifluoroacetate

[0204](2S)-1-[N-(tert-Butyloxycarbonyl)-S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile(130 mg, 0.29 mmol) was dissolved in trifluoroacetic acid (10 mL). After1 hour at room temperature the solvent was removed in vacuo to give awhite solid identified as(2S)-1-[S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitriletrifluoroacetate (116 mg,0.25 mmol, 86%).

[0205] [M+H]⁺=348.2

Example 13 3-[N^(ω)-(2-Quinoxaloyl)-L-lysinyl]thiazolidine Hydrochloride

[0206]

[0207] A.3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-quinoxaloyl)-L-lysinyl]thiazolidine

[0208] 3-[N^(α)-(tert-Butyloxycarbonyl)lysinyl]thiazolidine (128 mg, 0.4mmol) was dissolved in CH₂Cl₂ (10 mL). 2-Quinoxaloyl chloride (85 mg,0.44 mmol) and potassium carbonate (45.8 mg, 0.3 mmol) were added. Thereaction mixture was stirred at room temperature for 18 hours and thesolvent removed in vacuo. The residue was purified by flashchromatography (eluant: 99.5% chloroform, 0.5% methanol) to give acolourless oil identified as3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(2-quinoxaloyl)-L-lysinyl]thiazolidine(140 mg, 0.296 mmol, 74%).

[0209] B. 3-[N^(ω)-(2-Quinoxaloyl)-L-lysinyl]thiazolidine Hydrochloride

[0210]3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(2-quinoxaloyl)-L-lysinyl]thiazolidine(140 mg, 0.296 mmol) was dissolved in 4M HCl/dioxan (20 mL). After 1hour at room temperature the solvent was removed in vacuo to give awhite solid identified as3-[N^(ω)-(2-quinoxaloyl)-L-lysinyl]thiazolidine hydrochloride (128 mg,0.296 mmol, 100%).

[0211] [M+H]⁺=374.2

Example 14 3-[N^(ω)-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidineHydrochloride

[0212]

[0213] A.3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine

[0214] 3-Hydroxypyridine (14.9 mg, 0.138 mmol) was dissolved in CH₂Cl₂(20 mL). Phosgene (20% solution in toluene, 0.335 mL, 0.685 mmol) andpyridine (14 mg, 0.182 mmol) were added at 0° C. After 90 mins thesolvent was removed in vacuo and the residue dissolved in CH₂Cl₂ (20mL). 3-[N^(α)-(tert-Butyloxycarbonyl)ornithinyl]thiazolidine (42 mg,0.138 mmol) and triethylamine (28 mg, 0.28 mmol) were added. Thereaction mixture was stirred at room temperature for 18 hours and thesolvent removed in vacuo. The residue was purified by flashchromatography (eluant: 97% chloroform, 3% methanol) to give acolourless oil identified as3-[N^(α)-(tert-butyloxycarbonyl)-N^(ω)-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine(16 mg. 0.038 mmol, 27%).

[0215] B. 3-[N^(ω)-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidineHydrochloride

[0216]3-[N^(α)-(tert-Butyloxycarbonyl)-N^(ω)-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine(16 mg, 0.038 mmol) was dissolved in 4M HCl/dioxan (20 mL). After 1 hourat room temperature the solvent was removed in vacuo to give a whitesolid identified as3-[N^(ω)-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine hydrochloride(14 mg, 0.038 mmol, 100%).

[0217] [M+H]⁺=325.1

Example 15 3-[O-(3-Chlorobenzylcarbamoyl)serinyl]thiazolidineHydrochloride

[0218]

[0219] A. 3-[N-(tert-Butyloxycarbonyl)-L-serinyl]thiazolidine

[0220] N-(tert-Butyloxycarbonyl)-L-serine (2.1 g, 10.2 mmol) wasdissolved in CH₂Cl₂/DMF (9:1,50 mL). Thiazolidine (650 mg, 11.2 mmol),hydroxybenzotriazole(2.8 g, 20.7 mmol) and water soluble carbodiimide(3.9 g, 19.5 mmol) were added at 0° C. The pH was adjusted to pH8 withN-methylmorpholine. The reaction mixture was stirred at room temperaturefor 18 hours, the solvent removed in vacuo and the residue was taken upin ethyl acetate (150 mL). This solution was washed with 0.3M KHSO₄(1×50 mL), sat. NaHCO₃ (1×50 mL), water (1×50 mL) and brine (1×50 mL),dried (Na₂SO₄) and evaporated in vacuo to give a white solid identifiedas 3-[N-(tert-butyloxycarbonyl)-L-serinyl]thiazolidine (2.15 g, 7.78mmol, 76%).

[0221] B.3-[N-(tert-Butyloxycarbonyl)-O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine

[0222] 3-[N-(tert-Butyloxycarbonyl)-L-serinyl]thiazolidine (110 mg, 0.48mmol) was dissolved in DMF (10 mL) and 1,1′-carbonyl-diimidazole (71 mg,0.43 mmol) was added. After 2 hours at room temperature3-chlorobenzylamine (62 mg, 0.4 mmol) was added. After a further 18hours EtOAc (200 mL) was added. This solution was washed with 0.3M KHSO₄(1×50 mL), sat. NaHCO₃ (1×50 mL), water (4×50 mL) and brine (1×50 mL),dried (Na₂SO₄) and evaporated in vacuo to give a yellow oil. This waspurified by flash chromatography (eluant: 40% ethyl acetate, 60% pet.ether) to give a colourless oil identified as3-[N-(tert-butyloxycarbonyl)-O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine(158 mg, 0.36 mmol, 90%).

[0223] C. 3-[O-(3-Chlorobenzylcarbamoyl)-L-serinyl]thiazolidineHydrochloride

[0224]3-[N-(tert-Butyloxycarbonyl)-O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine(140 mg, 0.32 mmol) was dissolved in 4M HCl/dioxan (20 mL). After 1 hourat room temperature the solvent was removed in vacuo to give a whitesolid identified as3-[O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine hydrochloride (115mg, 0.3 mmol, 94%).

[0225] [M+H]⁺=344.1

[0226] The Examples set out in the following Tables were prepared byanalogous methods to the above. TABLE 1 Examples 16-162

Example n R⁸(CH₂)_(q) R¹ X¹ 16 17 18 19 1 2 1 2

CN CN H H CH₂CH₂S S 20 21 22 23 1 2 1 2

CN CN H H S S CH₂CH₂ 24 25 26 27 1 2 1 2

CN CN H H CH₂CH₂S S 28 29 30 31 1 2 1 2

CN CN H H CH₂CH₂S S 32 33 34 35 1 2 1 2

CN CN H H CH₂CH₂S S 36 37 38 1 2 1

CN CN H CH₂CH₂S 39 40 41 42 1 2 1 2

CN CN H H CH₂CH₂S S 43 44 45 46 1 2 1 2

CN CN H H CH₂CH₂S S 47 48 49 50 1 2 1 2

CN CN H H CH₂CH₂S S 51 52 53 54 1 2 1 2

CN CN H H CH₂CH₂S S 55 56 57 58 1 2 1 2

CN CN H H CH₂CH₂S S 59 60 61 62 1 2 1 2

CN CN H H CH₂CH₂S S 63 64 65 66 1 2 1 2

CN CN H H CH₂CH₂S S 67 68 69 70 1 2 1 2

CN CN H H CH₂CH₂S S 71 72 73 74 1 2 1 2

CN CN H H CH₂CH₂S S 75 76 77 78 1 2 1 2

CN CN H H CH₂CH₂S S 79 80 81 82 1 2 1 2

CN CN H H CH₂CH₂S S 83 84 85 86 1 2 1 2

CN CN H H CH₂CH₂S S 87 88 89 90 1 2 1 2

CN CN H H CH₂CH₂S S 91 92 93 94 1 2 1 2

CN CN H H CH₂CH₂S S 95 96 97 98 1 2 1 2

CN CN H H CH₂CH₂S S 99 100 101 102 1 2 1 2

CN CN H H CH₂CH₂S S 103 104 105 106 1 2 1 2

CN CN H H CH₂CH₂S S 107 108 109 110 1 2 1 2

CN CN H H CH₂CH₂S S 111 112 113 114 1 2 1 2

CN CN H H CH₂CH₂S S 115 116 117 118 1 2 1 2

CN CN H H CH₂CH₂S S 119 120 121 122 1 2 1 2

CN CN H H CH₂CH₂S S 123 124 125 126 1 2 1 2

CN CN H H CH₂CH₂S S 127 1

H S 128 129 1 2

H H S S 130 131 1 2

H H S S 132 1

H S 133 1

H S 134 1

H S 135 1

H S 136 1

H S 137 1

H S 138 1

H S 139 1

H S 140 1

H S 141 1

H S 142 1

H S 143 1

H S 144 1

H CH₂ 145 1

H CH₂ 146 1

H CH₂ 147 1

H CH₂ 148 1

H S 149 150 1 2

H H S S 151 151 1 2

H H S S 153 1

H S 154 1

H S 155 1

H S 156 1

H S 157 1

H S 158 1

H S 159 1

H S 160 1

H S 161 1

H S 162 1

H CH₂

[0227] TABLE 2 Examples 163-250

Example n R⁶R⁷N R¹ X¹ 163 164 165 166 1 2 1 2

CN CN H H CH₂CH₂S S 167 168 169 170 1 2 1 2

CN CN H H S S CH₂CH₂ 171 172 173 174 1 2 1 2

CN CN H H CH₂CH₂S S 175 176 177 178 1 2 1 2

CN CN H H CH₂CH₂S S 179 180 181 182 1 2 1 2

CN CN H H CH₂CH₂S S 183 184 185 186 1 2 1 2

CN CN H H CH₂CH₂S S 187 188 189 190 1 2 1 2

CN CN H H CH₂CH₂S S 191 1 NH₂ CN CH₂ 192 2 CN CH₂ 193 1 H S 194 2 H S195 196 197 198 1 2 1 2

CN CN H H CH₂CH₂S S 199 200 201 202 1 2 1 2

CN CN H H CH₂CH₂S S 203 204 205 206 1 2 1 2

CN CN H H CH₂CH₂S S 207 208 209 210 1 2 1 2

CN CN H H CH₂CH₂S S 211 212 213 214 1 2 1 2

CN CN H H CH₂CH₂S S 215 216 217 218 1 2 1 2

CN CN H H CH₂CH₂S S 219 220 221 222 1 2 1 2

CN CN H H CH₂CH₂S S 223 224 225 226 1 2 1 2

CN CN H H CH₂CH₂S S 227 228 229 230 1 2 1 2

CN CN H H CH₂CH₂S S 231 232 233 234 1 2 1 2

CN CN H H CH₂CH₂S S 235 236 237 238 1 2 1 2

CN CN H H CH₂CH₂S S 239 240 241 242 1 2 1 2

CN CN H H CH₂CH₂S S 243 244 245 246 1 2 1 2

CN CN H H CH₂CH₂S S 247 248 249 250 1 2 1 2

CN CN H H CH₂CH₂S S

[0228] TABLE 3 Examples 251-266

Example n R⁶R⁷N R¹ X¹ 251 252 253 254 1 2 1 2

CN CN H H CH₂CH₂S S 255 256 257 258 1 2 1 2

CN CN H H CH₂CH₂S S 259 260 261 262 1 2 1 2

CN CN H H CH₂CH₂S S 263 264 265 266 1 2 1 2

CN CN H H S S CH₂CH₂

[0229] TABLE 4 Examples 267-318

Example n R⁸(CH₂)_(q) R¹ X¹ 267 268 1 2

CN CN CH₂CH₂ 269 270 1 2

CN CN S S 271 272 1 2

CN CN CH₂CH₂ 273 274 1 2

CN CN CH₂CH₂ 275 276 1 2

CN CN CH₂CH₂ 277 278 1 2

CN CN CH₂CH₂ 279 280 1 2

CN CN CH₂CH₂ 281 282 1 2

H H CH₂CH₂ 283 284 1 2

H H CH₂CH₂ 285 286 1 2

H H CH₂CH₂ 287 288 1 2

H H S S 289 290 1 2

H H S S 291 292 1 2

H H S S 293 294 1 2

CN CN CH₂CH₂ 295 296 1 2

CN CN CH₂CH₂ 297 298 1 2

CN CN CH₂CH₂ 299 300 1 2

CN CN CH₂CH₂ 301 302 1 2

CN CN CH₂CH₂ 303 304 1 2

CN CN CH₂CH₂ 305 306 1 2

CN CN CH₂CH₂ 307 308 1 2

H H S S 309 310 1 2

H H S S 311 312 1 2

H H S S 313 314 1 2

H H S S 315 316 1 2

H H S S 317 318 1 2

H H S S

[0230] TABLE 5 Examples 319-378

Example R^(4B) R¹ X¹ 319 NH₂ CN CH₂ 320

CN CH₂ 321

CN CH₂ 322

CN CH₂ 323

CN CH₂ 324

CN CH₂ 325

CN CH₂ 326

CN CH₂ 327

H CH₂ 328

H CH₂ 329

H S 330

H S 331

H S 332

H S 333

CN CH₂ 334

CN CH₂ 335

CN S 336

CN S 337

CN CH₂ 338

CN CH₂ 339

CN CH₂ 340

CN CH₂ 341

H S 342

H S 343

H S 344

H S 345

H S 346

H S 347

H S 348

H S 349

H S 350

H S 351

H S 352

H S 353

H S 354

H S 355

H S 356

H S 357

H S 358

H S 359

H S 360

H S 361

H CH₂ 362

H CH₂ 363

H S 364

H S 365

H S 366

H S 367

H S 368

H S 369

H S 370

H S 371

H S 372

H S 373

H S 374

H S 375

H S 376

H CH₂ 377

H CH₂ 378

H CH₂

[0231] TABLE 6 Examples 379-418

Example n R⁸(CH₂)_(q) R¹ X¹ 379 380 1 2

CN CN CH₂CH₂ 381 382 1 2

CN CN CH₂CH₂ 383 384 1 2

CN CN CH₂CH₂ 385 386 1 2

CN CN CH₂CH₂ 387 388 1 2

CN CN CH₂CH₂ 389 390 1 2

CN CN CH₂CH₂ 391 392 1 2

CN CN CH₂CH₂ 393 394 1 2

CN CN CH₂CH₂ 395 396 1 2

H H CH₂CH₂ 397 398 1 2

H H S S 399 400 1 2

CN CN S S 401 402 1 2

CN CN CH₂CH₂ 403 404 1 2

CN CN CH₂CH₂ 405 406 1 2

CN CN CH₂CH₂ 407 408 1 2

CN CN CH₂CH₂ 409 410 1 2

CN CN CH₂CH₂ 411 412 1 2

CN CN CH₂CH₂ 413 414 1 2

H H S S 415 416 1 2

H H S S 417 418 1 2

H H S S

[0232] TABLE 7 Examples 419-438

Example m R⁶R⁷N 419 1

420 1

421 1

422 1

423 1

424 425 426 1 2 3

427 428 429 1 2 3

430 431 432 1 2 3

433 434 2 3

435 436 2 3

437 438 2 3

[0233] TABLE 8 Examples 439-450

Example n R⁸(CH₂)_(q) R¹ X¹ 439 440 1 2

CN CN CH₂CH₂ 441 442 1 2

CN CN CH₂CH₂ 443 444 1 2

H H S S 445 446 1 2

CN CN CH₂CH₂ 447 448 1 2

CN CN CH₂CH₂ 449 450 1 2

CN CN CH₂CH₂

Example 451 Determination of Activity

[0234] Compounds were assayed as inhibitors of DP-IV according to themethods described in WO95/15309. All the compounds described in theforegoing Examples were competitive inhibitors of DP-IV with K_(i)values less than 300 nM.

Example 452 Determination of Activity in Vivo

[0235] The anti-diabetic action of selected compounds was demonstratedin Zucker obese rats using a standard oral glucose tolerance test.Control rats were given a solution of glucose by oral gavage, and plasmaglucose levels were determined. These rats demonstrated a significanthyperglycaemia. Compounds according to the present invention weredissolved in glucose solution at various concentrations, such that therats could be given varying doses of the compound simultaneously withthe glucose challenge. The hyperglycaemic excursion was reduced in adose-dependent manner in animals receiving between 0.1 and 100 mg/kg ofDP-IV inhibitor.

Example 453 Pharmaceutical Formulation

[0236] Tablets containing 100 mg of the compound of Example 1 as theactive agent are prepared from the following: Compound of Example 1200.0 g Corn starch 71.0 g Hydroxypropylcellulose 18.0 gCarboxymethylcellulose calcium 13.0 g Magnesium stearate 3.0 g Lactose195.0 g Total 500.0 g

[0237] The materials are blended and then pressed to give 2000 tabletsof 250 mg, each containing 100 mg of the compound of Example 1.

[0238] The above demonstrates that the compounds according to thepresent invention are inhibitors of DP-IV and would accordingly beexpected to be useful as therapeutic agents for the treatment ofimpaired glucose tolerance, type II diabetes, and other diseases whereinhibition of this enzyme leads to an improvement in the underlyingpathology or the symptoms.

[0239] The present invention is further defined in the following Claims.

1. A compound selected from derivatives of formula 1, tautomers andstereoisomers thereof, and pharmaceutically acceptable salts of saidderivatives, tautomers and isomers

wherein: X¹ is selected from a sulphur atom, an oxygen atom, a sulphinylgroup, a sulphonyl group and a methylene group; X² is selected from O, Sand methylene; X³ is either NR⁵ or a carbonyl or thiocarbonyl group; R¹is either a hydrogen atom or a nitrile group; R² and R³ areindependently selected from H and C₁-C₆ alkyl, or together may be—(CH₂)_(p)—; R⁴ is R^(4A) when X³ is NR⁵ and R^(4B) when X³ is acarbonyl or thiocarbonyl group; R^(4A) is selected from R⁶R⁷NC(═O),R⁶R⁷NC(═S); R⁸(CH₂)_(q)C(═O), R⁸(CH₂)_(q)C(═S), R⁸(CH₂)_(q)SO₂,R⁸(CH₂)_(q)OC(═O) and R⁸(CH₂)_(q)OC(═S); R^(4B) is R⁶R⁷N; R⁵ is H orC₁-C₆ alkyl; R⁶ and R⁷ are selected independently from R⁸(CH₂)_(q) ortogether they are —(CH₂)₂—Z¹—(CH₂)₂— or —CHR⁹—Z²—CH₂—CHR¹⁰—; R⁸ isselected from H, alkyl, benzo-fused cycloalkyl, acyl, dialkylcarbamoyl,dialkylamino, N-alkylpiperidyl, optionally substituted aryl, optionallysubstituted α-alkylbenzyl, optionally substituted aroyl, optionallysubstituted arylsulphonyl and optionally substituted heteroaryl; R⁹ andR¹⁰ are selected independently from H, carbamoyl, hydroxymethyl andcyanomethyl; Z¹ is selected from a covalent bond, —(CH₂)_(r)—, —O—,—SO_(t)— and —N((CH₂)_(q)R⁸)—; Z² is an optionally substitutedortho-phenylene moiety; m is 1-3; n is 0-4; p is 2-5; q is 0-3; r is 1or 2; and t is 0-2; provided that when X² is CH₂, X³ is NH and R⁴ isR⁸CH₂O(CO) then R⁸ is not unsubstituted phenyl or nitrophenyl.
 2. Acompound according to claim 1 wherein R¹ is a nitrile group.
 3. Acompound according to claim 2 wherein the stereochemistry of the nitrilegroup is as shown in formula 2


4. A compound according to claim 1 or 2 wherein the stereochemistry ofthe centre adjacent to the primary amine is of the S configuration asshown in formula 3


5. A compound according to claim 4 wherein R¹ is a nitrile group and thestereochemistry of the nitrile group is as shown in formula 4


6. A compound according to any preceding claim wherein m is
 1. 7. Acompound according to any preceding claim wherein R² and R³ areindependently H or methyl.
 8. A compound according to any precedingclaim wherein m is 1, X² is —CH₂— and R² and R³ are both H.
 9. Acompound according to any of claims 1 to 7 wherein m is 1, X² is —O— andone of R² and R³ is methyl and the other is H.
 10. A compound accordingto any of claims 1 to 7 wherein m is 1, X² is —S— and R² and R³ are bothmethyl.
 11. A compound according to any preceding claim wherein X³ isNH.
 12. A compound according to claim 11 wherein m is 1, R² and R³ are Hand n is 1 or
 2. 13. A compound according to claim 12 wherein R⁴ is R⁸COor R⁸NHCO and R⁸ is optionally substituted heteroaryl.
 14. A compoundaccording to any of claims 1 to 7 wherein m is 1, X² is —CH₂—, R² and R³are both H, n is 0 and X³ is CO.
 15. A compound according to claim 14wherein R⁴ is R⁶NH.
 16. A compound according to claim 15 wherein R⁶ isoptionally substituted heteroaryl.
 17. A compound according to claim 13or claim 16 wherein the heteroaryl group is unsubstituted or mono- ordisubstituted and the substituents are selected from C₁-C₆ alkyl, C₁-C₆alkyloxy, fluoro, chloro and trifluoromethyl groups.
 18. A compoundaccording to claim 1 wherein R¹ is CN and X¹ is CH₂.
 19. A compoundaccording to claim 1 wherein R¹ is H and X¹ is S.
 20. A compoundaccording to claim 1 wherein X³ is NH and R⁴ is selected from R⁶R⁷N(CO),R⁸(CH₂)_(q)CO and R⁸(CH₂)_(q)SO₂.
 21. A compound according to claim 1selected from:(2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile,(2S)-1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrile,(2S)-1-[(2′S)-2′-Amino-4′-(pyrazinyl-2″-carbonylamino)butanoyl]pyrrolidine-2-carbonitrile,(4R)-3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile,(2S)-1-[N^(ω)-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile,1-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine,(2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile,3-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine,1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine,(2S)-1-[N^(ω)-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrle3-[N^(ω)-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine,3-[N^(ω)-(2-Quinoxaloyl)-L-lysinyl]thiazolidine,3-[N^(ω)-(2-Quinoxaloyl)-L-ornithinyl]thiazolidine,(2S)-1-[N^(ω)-(2-Quinoxaloyl)-L-ornithinyl]pyrrolidine-2-carbonitrile,3-[N^(ω)-(6-Methylpyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine,3-[N^(ω)-(Isoquinoline-3-carbonyl)-L-ornithinyl]thiazolidine,3-[N^(ω)-(6-Trifluoromethylnicotinoyl)-L-ornithinyl]thiazolidine,(2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrile,(2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile,3-[N^(ω)-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidine,3-[O-(3-Chlorobenzylcarbamoyl)serinyl]thiazolidine, and3-[(2′S)-2′-Amino-5′-oxo-5′-(1″,2″,3″,4″-tetrahydroisoquinolin-2″-yl)pentanoyl]-thiazolidine.22. A pharmaceutical composition for human therapeutic use comprising atleast one compound according to any preceding claim.
 23. A compositionfor the treatment of at least one of type 2 diabetes, impaired glucosetolerance, growth hormone deficiency, polycystic ovary syndrome, andauto-immune and inflammatory diseases, the composition comprising atleast one compound which is selected from derivatives of formula A,tautomers and stereoisomers thereof, and pharmaceutically acceptablesalts of said derivatives, tautomers and isomers, or which is accordingto any of claims 2 to 21

wherein: X¹ is selected from a sulphur atom, an oxygen atom, a sulphinylgroup, a sulphonyl group and a methylene group; X² is selected from O, Sand methylene; X³ is either NR⁵ or a carbonyl or thiocarbonyl group; R¹is either a hydrogen atom or a nitrile group; R² and R³ areindependently selected from H and C₁-C₆ alkyl, or together may be—(CH₂)_(p)—; R⁴ is R^(4A) when X³ is NR⁵ and R^(4B) when X³ is acarbonyl or thiocarbonyl group; R^(4A) is selected from R⁶R⁷NC(═O),R⁶R⁷NC(═S); R⁸(CH₂)_(q)C(═O), R⁸(CH₂)_(q)C(═S), R⁸(CH₂)_(q)SO₂,R⁸(CH₂)_(q)OC(═O) and R⁸(CH₂)_(q)OC(═S); R^(4B) is R⁶R⁷N; R⁵ is H orC₁-C₆ alkyl; R⁶ and R⁷ are selected independently from R⁸(CH₂)_(q) ortogether they are —(CH₂)₂—Z¹—(CH₂)₂— or —CHR⁹—Z²—CH₂—CHR¹⁰—; R⁸ isselected from H, alkyl, benzo-fused cycloalkyl, acyl, dialkylcarbamoyl,dialkylamino, N-alkylpiperidyl, optionally substituted aryl, optionallysubstituted α-alkylbenzyl, optionally substituted aroyl, optionallysubstituted arylsulphonyl and optionally substituted heteroaryl; R⁹ andR¹⁰ are selected independently from H, carbamoyl, hydroxymethyl andcyanomethyl; Z¹ is selected from a covalent bond, —(CH₂)_(r)—, —O—,—SO_(t)— and —N((CH₂)_(q)R⁸)—; Z² is an optionally substitutedortho-phenylene moiety; m is 1-3; n is 0-4; p is 2-5; q is 0-3; r is 1or 2; and t is 0-2.
 24. The use of a compound for the preparation of apharmaceutical composition for the treatment of at least one of type 2diabetes, impaired glucose tolerance, growth hormone deficiency,polycystic ovary syndrome, and auto-immune and inflammatory diseases,the compound being selected from derivatives of formula A, tautomers andstereoisomers thereof, and pharmaceutically acceptable salts of saidderivatives, tautomers and isomers, or being according to any of claims2 to 21

wherein: X¹ is selected from a sulphur atom, an oxygen atom, a sulphinylgroup, a sulphonyl group and a methylene group; X² is selected from O, Sand methylene; X³ is either NR⁵ or a carbonyl or thiocarbonyl group; R¹is either a hydrogen atom or a nitrite group; R² and R³ areindependently selected from H and C₁-C₆ alkyl, or together may be—(CH₂)_(p)—; R⁴ is R^(4A) when X³ is NR⁵ and R^(4B) when X³ is acarbonyl or thiocarbonyl group; R^(4A) is selected from R⁶R⁷NC(═O),R⁶R⁷NC(═S); R⁸(CH₂)_(q)C(═O), R⁸(CH₂)_(q)C(═S), R⁸(CH₂)_(q)SO₂,R⁸(CH₂)_(q)OC(═O) and R⁸(CH₂)_(q)OC(═S); R^(4B) is R⁶R⁷N; R⁵ is H orC₁-C₆ alkyl; R⁶ and R⁷ are selected independently from R⁸(CH₂)_(q) ortogether they are —(CH₂)₂—Z¹—(CH₂)₂— or —CHR⁹—Z²—CH₂—CHR¹⁰—; R⁸ isselected from H, alkyl, benzo-fused cycloalkyl, acyl, dialkylcarbamoyl,dialkylamino, N-alkylpiperidyl, optionally substituted aryl, optionallysubstituted α-alkylbenzyl, optionally substituted aroyl, optionallysubstituted arylsulphonyl and optionally substituted heteroaryl; R⁹ andR¹⁰ are selected independently from H, carbamoyl, hydroxymethyl andcyanomethyl; Z¹ is selected from a covalent bond, —(CH₂)_(r)—, —O—,—SO_(t)— and —N((CH₂)_(q)R⁸)—; Z² is an optionally substitutedortho-phenylene moiety; m is 1-3; n is 0-4; p is 2-5; q is 0-3; r is 1or 2; and t is 0-2.
 25. The use of a compound for the treatment of type2 diabetes, impaired glucose tolerance, growth hormone deficiency,polycystic ovary syndrome, or auto-immune or inflammatory disease, thecompound being selected from derivatives of formula A, tautomers andstereoisomers thereof, and pharmaceutically acceptable salts of saidderivatives, tautomers and isomers, or being according to any of claims2 to 21

wherein: X¹ is selected from a sulphur atom, an oxygen atom, a sulphinylgroup, a sulphonyl group and a methylene group; X² is selected from O, Sand methylene; X³ is either NR⁵ or a carbonyl or thiocarbonyl group; R¹is either a hydrogen atom or a nitrile group; R² and R³ areindependently selected from H and C₁-C₆ alkyl, or together may be—(CH₂)_(p)—; R⁴ is R^(4A) when X³ is NR⁵ and R^(4B) when X³ is acarbonyl or thiocarbonyl group; R^(4A) is selected from R⁶R⁷NC(═O),R⁶R⁷NC(═S); R⁸(CH₂)_(q)C(═O), R⁸(CH₂)_(q)C(═S), R⁸(CH₂)_(q)SO₂,R⁸(CH₂)_(q)OC(═O) and R⁸(CH₂)_(q)OC(═S); R^(4B) is R⁶R⁷N; R⁵ is H orC₁-C₆ alkyl; R⁶ and R⁷ are selected independently from R⁸(CH₂)_(q) ortogether they are —(CH₂)₂—Z¹—(CH₂)₂— or —CHR⁹—Z²—CH₂—CHR¹⁰—; R⁸ isselected from H, alkyl, benzo-fused cycloalkyl, acyl, dialkylcarbamoyl,dialkylamino, N-alkylpiperidyl, optionally substituted aryl, optionallysubstituted α-alkylbenzyl, optionally substituted aroyl, optionallysubstituted arylsulphonyl and optionally substituted heteroaryl; R⁹ andR¹⁰ are selected independently from H, carbamoyl, hydroxymethyl andcyanomethyl; Z¹ is selected from a covalent bond, —(CH₂)_(r)—, O—,—SO_(t)— and —N((CH₂)_(q)R⁸)—; Z² is an optionally substitutedortho-phenylene moiety; m is 1-3; n is 0-4; p is 2-5; q is 0-3; r is 1or 2; and t is 0-2.
 26. A method of treatment of at least one of type 2diabetes, impaired glucose tolerance, growth hormone deficiency,polycystic ovary syndrome, and auto-immune and inflammatory diseases,which comprises the administration to a patient in need of suchtreatment of a therapeutically effective amount of at least one compoundwhich is selected from derivatives of formula A, tautomers andstereoisomers thereof, and pharmaceutically acceptable salts of saidderivatives, tautomers and isomers, or which is according to any ofclaims 2 to 21

wherein: X¹ is selected from a sulphur atom, an oxygen atom, a sulphinylgroup, a sulphonyl group and a methylene group; X² is selected from O, Sand methylene; X³ is either NR⁵ or a carbonyl or thiocarbonyl group; R¹is either a hydrogen atom or a nitrile group; R² and R³ areindependently selected from H and C₁-C₆ alkyl, or together may be—(CH₂)_(p)—; R⁴ is R^(4A) when X³ is NR⁵ and R^(4B) when X³ is acarbonyl or thiocarbonyl group; R^(4A) is selected from R⁶R⁷NC(═O),R⁶R⁷NC(═S); R⁸(CH₂)_(q)C(═O), R⁸(CH₂)_(q)C(═S), R⁸(CH₂)_(q)SO₂,R⁸(CH₂)_(q)OC(═O) and R⁸(CH₂)_(q)OC(═S); R^(4B) is R⁶R⁷N; R⁵ is H orC₁-C₆ alkyl; R⁶ and R⁷ are selected independently from R⁸(CH₂)_(q) ortogether they are —(CH₂)₂—Z¹—(CH₂)₂— or —CHR⁹—Z²—CH₂—CHR¹⁰—; R⁸ isselected from H, alkyl, benzo-fused cycloalkyl, acyl, dialkylcarbamoyl,dialkylamino, N-alkylpiperidyl, optionally substituted aryl, optionallysubstituted α-alkylbenzyl, optionally substituted aroyl, optionallysubstituted arylsulphonyl and optionally substituted heteroaryl; R⁹ andR¹⁰ are selected independently from H, carbamoyl, hydroxymethyl andcyanomethyl; Z¹ is selected from a covalent bond, —(CH₂)_(r)—, —O—,—SO_(t)— and —N((CH₂)_(q)R⁸)—; Z² is an optionally substitutedortho-phenylene moiety; m is 1-3; n is 0-4; p is 2-5; q is 0-3; r is 1or 2; and t is 0-2.